The accurate diagnosis of thyroid nodules continues to challenge physicians managing patients with thyroid disease. Patients with cytologically indeterminate nodules are often referred for diagnostic surgery, though most of these nodules prove post-surgery to be benign. This limitation of FNA cytology in the pre-operative diagnosis leads to a clinical need for reliable pre-operative molecular markers to distinguish benign from malignant thyroid nodules. MicroRNAs (miRs) are an important class of regulatory RNAs, which have a profound impact on a wide array of biological processes. These small (typically 18-24 nucleotides long) non-coding RNA molecules can modulate protein expression pattern by promoting RNA degradation, inhibiting mRNA translation, and also by affecting gene transcription. miRs play pivotal roles in diverse processes such as development and differentiation, control of cell proliferation, stress response and metabolism. The expression of many miRs was found to be altered in numerous types of human cancer, and in some cases suggesting that such alterations may play a causative role in tumor progression.
The thyroid gland is formed of two main types of cells: the follicular cells and the C or parafollicular cells. Follicular cells produce thyroid hormones, which are regulators of human metabolism. Overproduction of thyroid hormone (hyperthyroidism) causes rapid or irregular heartbeat, trouble sleeping, nervousness, hunger, weight loss, and a feeling of being too warm. In counterpart, hypothyroidism causes metabolism slowdown, tiredness, and weight gain. Thyroid hormone release is regulated by the thyroid-stimulating hormone (TSH), produced by the pituitary gland. The C cells produce calcitonin, a hormone responsible for use of calcium. Lymphocytes and stromal cells are also found in the thyroid.
Thyroid cancer is the eighth most common cancer in the United States, and the most rapidly increasing cancer in the US, with more than 60,000 new cases diagnosed every year, and being the cause of about 1,800 deaths in 2014. Thyroid cancer usually presents itself as a palpable thyroid nodule. Different types of thyroid tumors develop from different cell types, which is a determinant for the gravity and the optimal treatment administered. Most of the growths and tumors in the thyroid gland are benign (non-cancerous) but others are malignant (cancerous).
Approximately 95% of thyroid cancers are differentiated thyroid carcinomas (DTC) that arise from thyroid follicular cells. There are two histological subtypes of DTC: papillary thyroid carcinoma (PTC) type (90-95%) and follicular thyroid carcinoma (FTC) type (5-10%).
The most commonly used method for thyroid cancer diagnosis is biopsy by fine-needle aspiration (FNA). FNA samples are routinely examined for cytology to determine whether the nodules are benign or cancerous. The sensitivity and specificity of the cytological examination of an FNA sample range from 68% to 98%, and 72% to 100%, respectively, depending on institutions and doctors. Unfortunately, in at least 25% of the cases the FNA specimens collected are either inadequate for diagnosis or indeterminable by cytology. In current medical practice, most patients with indeterminate results undergo surgery, and are subject to all risks and consequences of the surgical procedure. Follow-up results show that only 25% of the patients operated on are diagnosed with cancer, meaning that 75% of the patients underwent an unnecessary surgical procedure. Surgery entails significant cost and morbidity. One study has shown that adding molecular testing could have an overall positive impact on healthcare cost and patients' quality of life, reaching up to 74% fewer surgeries for benign nodules with no greater number of untreated cancers. Over a 5-year period, the study estimated a savings of almost $1,500 per patient [Li et al. 2011 J Clin Endocrinol. Metab 96(11): E1719-E1726].
When examining cytochemical or genetic markers, there is no unique marker that on its own is able to provide reliable results in order to replace the morphologic diagnosis of thyroid lesions. U.S. Pat. No. 7,319,011 describes the measuring the expression of any one of the genes DDIT3, ARG2, ITM1, C1orf24, TARSH, and ACO1 in a test follicular thyroid specimen for distinguishing between follicular adenoma (FA) from follicular carcinoma (FC). U.S. Pat. No. 7,670,775 describes the analysis of the expression of CCND2, PCSK2, and PLAB for identifying malignant thyroid tissue. U.S. Pat. No. 6,723,506 describes the molecular characterization of PAX8-PPAR1 molecules in connection with diagnosis and treatment of thyroid follicular carcinomas. U.S. Pat. No. 7,378,233 describes the occurrence of the T1796A mutation of the BRAF gene in 24 (69%) of papillary thyroid carcinomas.
Accumulated efforts have been invested in finding a molecular diagnostic test which will overcome the uncertainty of indeterminate cytology, and ultimately eliminate unnecessary surgery for non-cancer patients [Chen, Y. T. et. al. (2008) Mod. Pathol. 21, 1139-1146; He, H. et al. (2005) Proc. Natl Acad. Sci. USA 102, 19075-19080; Nikiforova, M. N. et al. (2009) Endocr. Pathol. 20, 85-91; Pallante, P. et al. (2006) Endocr. Relat. Cancer 13, 497-508; Nikiforova, M. N. et al. (2008) J. Clin. Endocrinol. Metab. 93, 1600-1608; Visone, R. et al. (2007) Endocr. Relat. Cancer 14(3):791-8; US 2014/0030714 A1; U.S. Pat. No. 8,541,170; US 2012/0220474 A1; U.S. Pat. No. 8,465,914; U.S. Pat. No. 7,598,052; U.S. Pat. No. 8,202,692; WO 2013/066678; WO 2012/129378; US 2013/0237590; EP 2772 550 A1; Pallante et al. (2010) Endocrine-Related Cancer 17 F91-F104; Dettmer et al. (2014) J Mol Endocrinol. March 6; 52(2):181-9].
Nonetheless, numerous are the challenges that remain. It is of great necessity to develop a molecular assay with not only high sensitivity and specificity, but also that is able to deal with samples that failed the cytology analysis and that fall under the category of indeterminate samples. The present invention provides solutions for this challenge.